Colorectal neoplasia prophylaxis

ABSTRACT

Development of colorectal neoplasia in a patient subject or predisposed to colorectal neoplasia is reduced by the steps of (a) determining a patient is subject or predisposed to colorectal neoplasia; and (b) enterically delivering into the gut of the person an effective amount of an aminoglycoside antibiotic having poor gut absorption, whereby the development of the colorectal neoplasia is reduced as compared with otherwise similar non-treated patients.

FIELD OF THE INVENTION

The field of this invention is use of aminoglycoside antibiotics toreduce development of colorectal neoplasia.

BACKGROUND OF THE INVENTION

Aminoglycosides are potent bactericidal antibiotics, particularly activeagainst aerobic, gram-negative bacteria and act synergistically againstcertain gram-positive organisms. Gentamicin is the most commonly usedaminoglycoside, but amikacin may be particularly effective againstresistant organisms. Aminoglycosides are used in the treatment of severeinfections of the abdomen and urinary tract, as well as bacteremia andendocarditis. Aminoglycosides are poorly absorbed from thegastrointestinal tract. After parenteral administration, aminoglycosidesare primarily distributed within the extracellular fluid. Penetration ofbiologic membranes is poor because of the drug's polar structure, andintracellular concentrations are usually low, with the exception of theproximal renal tubule; for review, see, Gonzalez et al., American FamilyPhysician, Nov. 15, 1998; The Merck Manual of Diagnosis and Therapy,1999, 17^(th) Ed, Chap. 153.

Hu (1998, PNAS 95, 9791-795) and Hu et al. (U.S. Pat. No. 6,482,802)report that neomycin was the only member of a panel of aminoglycosideantibiotics that was able to reduce angiogenin-induced cellproliferation and angiogenesis, as measured using cultured humanendothelial cells and chick embryo chorioallantoic membranes (CAM).Streptomycin, kanamycin, gentamicin, amikacin, and paromomycin werefound to lack anti-angiogenic activity. This Hu attributes tophospholipase C (PLC) inhibition because neomycin is the onlyaminoglycoside antibiotic of the panel tested that inhibits PLC, andbecause U-73122, another PLC inhibitor, similarly inhibitedangiogenin-induced cell proliferation and angiogenesis. See abstract ofHu paper; col. 20, lines 22-28, col. 21, line 66—col. 22, line 8, andcol. 27, lines 8-27 of Hu patent. The Hu patent also reports thatinjected neomycin was able to reduce growth of culturedangiogenin-secreting tumor cells (Olson et al., Int J Cancer. 2002 Apr.20;98:923-9; Clin Cancer Res. 2001 November;7:3598-605) transplantedinto mice; see, col. 27, line 43—col. 29, line 10 of Hu patent.

From these findings, Hu suggests use of neomycin for treating anyangiogenin-induced angiogenesis. The Hu patent purports applicability toall angiogenesis-related diseases, which are “myriad and varied” and“include, but are not limited to, various forms of neovascularization orhypervascularization diseases, inflammatory diseases, arthritis andcancer”, and: then goes on to recite a laundry-list of all major solidand blood-borne tumors, corneal and retinal diseases, inflammatorydiseases, and various infectious diseases, including AIDS (col. 16, line56—col. 18, line 16). Among the 60+ recited solid tumors is colon cancer(col. 17, lines 10-11).

Hu does not provide the public with any cure for cancer, inflammatorydisease, arthritis, or AIDS. He has shown that neomycin can reduceangiogenin-induced angiogenesis and growth in CAM and cultured cellmodels, and from these data, claims to be able to treat innumerablehuman diseases. In fact, anti-angiogenesis drugs have notoriously failedto live up to their promise as cancer therapies, primarily because thereare dozens of alternative factors which can drive angiogenesis. ScienceJournal, WSJ, Jul. 11, 2003. Furthermore, reports using azoxymethane(AOM) and 1,2-dimethyl hydrazine (DMH)-induced colon cancers havesuggested neomycin is not a viable therapy for colon cancer, and mayactually increase the incidence of colon adenocarcinomas (Reddy et al.,1984 JNCI 73, 275-9; Panda et al., 1999, Br J Cancer 80, 1132-6).

With the recent development of colon cancer animal models based ondefined genetic lesions (e.g. Zhu et al., Cell 1998, 94, 703-714), theuse of chemical carcinogenesis models like AOM and DMH has become lessfavored (Boivin et al., Gastroenterology 2003, 124, 762-777). Hence, arecent review of colon cancer chemoprevention surveys the many agentsreported to influence intestinal tumors, and does not include anyantibiotics; see, Table 1 of Corpet et al., Cancer Epidemiology,Biomakers & Prevention 2003, 12, 391-400. Work in colon cancer animalmodels has suggested that intestinal neoplasia is independent of gutmicrobial status (Dove et al., Cancer Res 1997, 57, 812-14).

The present inventors made the serendipitous discovery that entericallydelivered aminoglycoside antibiotics can dramatically reduce thedevelopment of colorectal carcinogenesis in defined rodent models. Theinventors here clinically extend these animal model findings by showingthat enterically delivered aminoglycoside antibiotics can inhibit largebowel carcinogenesis in human patients with familial adenomatouspolyposis. The inventors further extend these findings by showing thataltering the population profile of gut flora, through a defined regimenof aminoglycoside antibiotic treatment and supplementation of non-targetgut microbes such as Lactobacillus, is effective in preventing theformation of colorectal polyps and colorectal cancer. The inventorsdemonstrate prevention of spontaneous tumor formation indicating thatthe disclosed protocols interfere with early processes of carcinogenesisthat are distinct from angiogenesis.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for reducing developmentof colorectal neoplasia in a patients subject or predisposed tocolorectal neoplasia The general methods comprise the steps of (a)determining a patient is subject or predisposed to colorectal neoplasia;and (b) enterically delivering into the gut of the person an effectiveamount of an aminoglycoside antibiotic having poor gut absorption,whereby the development of the colorectal neoplasia is reduced ascompared with otherwise similar non-treated patients.

Colorectal neoplasias encompass large intestine, bowel, colon and rectalneoplasias, including colorectal neoplasias that are non-angiogeninsecreting and/or non-angiogenin dependent. Preferred target patientshave undergone removal or ablation of a colorectal neoplasia and aredetermined to be predisposed to colorectal neoplasia recurrence,including patients having a known predisposition, particularly a geneticpredisposition, due to familial history of colon cancer, especially inassociation with a known syndrome such as familial adenomatous polyposisor hereditary non-polyposis colon cancer. Hence, the determining stepmay comprise detecting an indication of or predisposition to polyps orcolorectal cancer.

The recited aminoglycoside antibiotic may be one of a plurality ofdifferent antibiotics, particularly different aminoglycosideantibiotics, and the delivering step may be effected by delivering theantibiotics in periodic dosages of different subsets of the antibiotics.The methods may further comprise introducing into the gut an effectiveamount of a probiotic, gut-beneficial microbial culture, such as speciesof Lactobacillus, Bifidobacteria, Bacteroides, Streptococcus, andSaccharomyces.

The invention also provides kits specifically tailored for the subjectmethods, for delivering an aminoglycoside antibiotic having poor gutabsorption. Typically, such kits comprise an aminoglycoside antibiotichaving poor gut absorption, and an associated instructional mediumdescribing use of the antibiotic in a subject method. The kits mayinclude varying doses of the aminoglycoside antibiotic, alone or incombination with another antibiotic or a defined dose of a probiotic,gut-beneficial microbe such as Lactobacillus.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

The invention provides methods and compositions for reducing developmentof colorectal neoplasia in patients, particularly patients known to besubject or predisposed to colorectal neoplasia. The general methodscomprise the steps of (a) determining a patient is subject orpredisposed to colorectal neoplasia; and (b) enterically delivering intothe gut of the person an effective amount of an aminoglycosideantibiotic having poor gut absorption, whereby the development of thecolorectal neoplasia is reduced as compared with otherwise similarnon-treated patients.

Colorectal neoplasias of the invention include neoplasias of the colon,bowel, rectum or large intestine, particularly adenomatous polyps andcolon carcinoma. In particular embodiments, the colorectal neoplasia isnon-angiogenin secreting and/or non-angiogenin dependent. In particularembodiments, the subject patient has undergone removal or ablation of acolorectal neoplasia and is determined to be predisposed to colorectalneoplasia recurrence, including genetically predisposed to thedevelopment of colorectal neoplasia.

Preferred target patients are predetermined to have a personal orfamilial history of colorectal neoplasia, or are predetermined to be atrelatively high risk of developing a colorectal neoplasia. Hence, thedetermining step may comprise detecting an indication of orpredisposition to polyps or colorectal cancer. In a particularembodiment, the invention reduces the risk of reccurrence of colorectalneoplasia.

Exemplary aminoglycoside antibiotics useful in the invention includeAmikacin (Amikin®), Gentamicin (Garamycin®), Kanamycin (Kantrex®),Neomycin (Mycifradin®), Netilmicin (Netromycin®), Paromomycin(Humatin®), Streptomycin, and Tobramycin (TOBI Solution®, TobraDex®,Nebcin®). However, the methods may be practiced with alternativeaminoglycosides which function similarly, which is readily determinedempirically, such as in the animal models described below. In aparticular embodiment, the administered aminoglycoside antibiotic isother than neomycin.

In a particular embodiment, the aminoglycoside antibiotic is one of aplurality of different antibiotics, preferably including or beingdifferent aminoglycoside antibiotics, and the delivering step iseffected by delivering the antibiotics in periodic dosages of differentsubsets of the antibiotics. Optimal dosages are readily determinedempirically as exemplified below and the invention has been documentedwith numerous example of known safe and intestinally-effective oraldosages of subject antibiotics.

The invention has been documented with a variety of aminoglycosideantibiotics, however alternative antibiotics may be substituted in thesame methods so long as they provide the requisite reduction in thedevelopment of colorectal cancer, assayed as described below, andpreferably provide poor gut absorption, similar to aminoglycosideantibiotics, such as neomycin. Furthermore, methods for modifying ordelivering otherwise highly gut absorbable antibiotics to or in a poorlygut absorbable form are known in the art, and include gut-retainingchemical moieties, chelators, enteric coatings, etc.; see, Remington'sPharmaceutical Science, Mack Publishing Co, NJ (1991). In addition,suitable excipients or carriers and methods for preparing administrablecompositions are known or apparent to those skilled in the art and aredescribed in more detail in publications such as Remington's.

The invention also provides embodiments which forestall or help avoiddevelopment of undesirable effects of antibiotic usage, such asside-effects, antibiotic resistance, etc. These embodiments adoptestablished procedures for minimizing development of side effects,resistance, etc. For example, administered antibiotics may beadministered in punctuated and/or sequential dosage regimes. In aparticular embodiment, a plurality of different antibiotics areadministered in sequential dosage regimes, alternating exposures to eachantibiotic over time. Hence, the antibiotics may be provided in a cyclicdispenser and/or with instructions providing a dosage regime of a firstantibiotic or cocktail, followed by a second dosage regime of a second,different antibiotic or cocktail, etc. In a particular embodiment, thedispensers are designed like conventional birth control pill dispensers,wherein the composition of the pills differs over time and they aretaken in a specific sequence for a defined period, after which the cyclejust starts over again.

In particular embodiments, the invention further comprises introducinginto the gut an effective amount of a probiotic, gut-beneficialmicrobial culture. A wide range of gut-beneficial microbes are known inthe art, including species of Lactobacillus, Bifidobacteria,Bacteroides, Streptococcus, and Saccharomyces. Exemplary well-knownspecies include L. acidophilus, L. reuteri, L. acidophilus, L.bulgaricus, L. plantarum, L. casei, L. gasseri, L. GG, S. thermophilus,S. salivarius, B. bifidus, and Saccharomyces boulardii. The probioticmicrobes are typically administered orally, typically in a defineddosage regime. The microbe may be one of a plurality of differentmicrobes, and the delivering step is effected by delivering the microbesin periodic dosages of different subsets of the antibiotics, or incocktails or mixtures of different sets or subsets of various microbes.For example, in one embodiment dosages are provides by capsulescontaining a combination of L. fermentum RC-14 and L. rhamnosus GR-1and/or L. rhamnosus GG in freeze-dried form (a total of >10⁹ viablebacteria). The probiotics may be administered in the context of aperiodic or changing antibiotic dosage regime, such as described above.

The invention also provides kits specifically tailored to practicing thesubject methods. In one embodiment, the kits are constructed fordelivering an aminoglycoside antibiotic having poor gut absorption, andcomprise an aminoglycoside antibiotic having poor gut absorption,typically in premeasured, defined dosages, and an associatedinstructional medium describing use of the antibiotic in a subjectmethod.

EXAMPLES

In the following examples, we demonstrate that orally-deliveredaminoglycosides reduce development of colorectal neoplasia.

I. Orally-delivered aminoglycosides reduce development of colon cancerin animal models predisposed to colon neoplasia. Smad3 mutant andAPC/MIN mice are fed on a standard laboratory diet, with aminoglycosideantibiotics added to the water. For example, 4 ml neomycin sulfate(BIOSOL, Pharmacia & Upjohn Company) was diluted in 500 ml steriledrinking water to give a final concentration of 1.6 mg/ml neomycinsulfate (1.1 mg/ml neomycin). Metronidazole was diluted in steriledrinking water to a final concentration of 0.5 mg/ml. The sameconcentrations are used for either combined or separate drug trials.

Smad3 mutant mice are maintained in 129Sv strain; APC/MIN mice aremaintained in C57BL/6J. Both Smad3 mutant and APC/MIN mice are startedon antibiotics at 5 weeks of age (range: 4-6 weeks). Mice are monitoredand scored for survival and visible rectal tumors. Mice are sacrificedwhen showing severe distress (typically rectal prolapse with significantdehydration,. hypokinesis, cachexia). At sacrifice mice are dissectedand their overall state assessed. Large intestines were harvested,opened longitudinally, and tumor number, location, and size recorded.Note that Smad3 mutant mice comprise an operable neo-resistance gene;hence, any neomycin absorbed from their gut should be inactivated.

Approximately 90% of control Smad3 mutant mice develop colorectal cancerwithin seven months. However, orally delivered aminoglycosides(amikacin, gentamicin, kanamycin, netilmicin, paromomycin, streptomycin,and tobramycin) prevent tumor development in over 90% of our Smad3 mice.For example, 9 of 10 untreated mice developed colorectal tumors within24 weeks (i.e., by the age of 24 weeks). Oral treatment with neomycinhowever, prevented the development of tumors in 11 of 12 mice at 24weeks. We find similar reductions in colon cancer development across thetested aminoglycosides in both Smad3 mutant and APC/MIN mice. Incontrast, a metronidazole treatment group was not significantlydifferent from non-treated controls.

II. Orally-delivered aminoglycosides reduce development of colorectalpolyps in patients with familial adenomatous polyposis. In this study wedemonstrate the efficacy of aminoglycoside antibiotices on colorectalpolyps in patients with familial adenomatous polyposis. We adapted thedouble-blind, placebo-controlled study format of Steinbach et al., 2000,NEJM, 342:1946-1952 to randomly assign patients to one of four treatmentgroups (neomycin or paramomycin, at 1 or 2 g daily in orallyadministered capsules) or a placebo group for six months. All patientsundergo endoscopy at the beginning and end of the study. The number andsize of polyps is determined from photographs and videotapes; theresponse to treatment is expressed as the mean percent change from baseline.

Results: At base line, the mean (±SD) number of polyps in focal areaswhere polyps are counted is similar (about 10±10) in patients assignedto placebo or treatment groups. After six months, the patients receiving1 or 2 g/day of either neomycin or paramomycin have about a 30 percentreduction in the mean number of colorectal polyps and a 30 percentreduction in the polyp burden (the sum of polyp diameters), whereas theplacebo group does not exhibit a: significant reduction. The improvementin the extent of colorectal polyposis in the treatment groups isconfirmed by a panel of endoscopists who independently review thevideotapes. The incidence of adverse events was similar among thegroups.

Conclusion: In patients with familial adenomatous polyposis, six monthsof daily treatment with 1 or 2 g of either neomycin or paramomycin leadsto a significant reduction in the number of colorectal polyps.

III. Orally-delivered aminoglycosides reduce development of colorectaladenomas in patients with previous colorectal cancer. In this study, wedemonstrate the efficacy of orally-delivered aminoglycosides on theincidence of colorectal adenomas. We adapted the double-blind,placebo-controlled trial format of Sandier, et al. 2003, NEJM 348,883-90 to randomly assign patients with prior colorectal cancer to oneof four treatment groups (neomycin or paramomycin, at 1 or 2 g daily inorally administered capsules) or a placebo group for six months. Wedetermine the proportion of patients with adenomas, the number ofrecurrent adenomas, and the time to the development of adenoma betweenrandomization and subsequent colonoscopic examinations. Relative risksare adjusted for age, sex, cancer stage, the number of colonoscopicexaminations, and the time to first colonoscopy.

Assigned patients have had at least one colonoscopic examination amedian of 12 months after randomization. One or more adenomas are foundin approximately 10 percent of patients in the four treatment groups andabout 30 percent of patients in the placebo group. The mean number ofadenomas is also lower in all four treatment groups than the placebogroup. Hence, the average, adjusted relative risk of any recurrentadenoma in the treatment groups, as compared with the placebo group, wasabout 0.5. The time to the detection of a first adenoma is also longerin each treatment group than in the placebo group.

Conclusion: We find that daily use of 1 or 2 g of either neomycin orparamomycin decreases the occurrence of new colorectal adenomas amongpatients with a history of colorectal cancer.

The foregoing descriptions of particular embodiments and examples areoffered by way of illustration and not by way of limitation. Unlesscontraindicated or noted otherwise, in these descriptions and throughoutthis specification, the terms “a” and “an” mean one or more, the term“or” means and/or.

All publications and patent applications cited in this specification andall references cited therein are herein incorporated by reference as ifeach individual publication or patent application or reference werespecifically and individually indicated to be incorporated by reference.Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be readily apparent to those of ordinary skill inthe art in light of the teachings of this invention that certain changesand modifications may be made thereto without departing from the spiritor scope of the appended claims.

1. A method of reducing development of colorectal neoplasia in a patientsubject or predisposed to colorectal neoplasia, the method comprisingthe steps of: determining a patient is subject or predisposed tocolorectal neoplasia; and enterically delivering into the gut of theperson an effective amount of an aminoglycoside antibiotic having poorgut absorption, whereby the development of the colorectal neoplasia isreduced as compared with otherwise similar non-treated patients.
 2. Themethod of claim 1, wherein the colorectal neoplasia is non-angiogeninsecreting or non-angiogenin dependent.
 3. The method of claim 1, whereinthe colorectal neoplasia is adenomatous polyps.
 4. The method of claim1, wherein the colorectal neoplasia is hereditary non-polyposis coloncancer.
 5. The method of claim 1, wherein the patient has undergoneremoval or ablation of a colorectal neoplasia and is determined to bepredisposed to colorectal neoplasia recurrence.
 6. The method of claim1, wherein the aminoglycoside antibiotic is selected from the groupconsisting of: Amikacin (Amikin®), Gentamicin (Garamycin®), Kanamycin(Kantrex®), Neomycin (Mycifradin®), Netilmicin (Netromycin®),Paromomycin (Humatin®), Streptomycin, and Tobramycin (TOBI Solution®,TobraDex®, Nebcin®).
 7. The method of claim 1, wherein theaminoglycoside antibiotic is other than neomycin.
 8. The method of claim1, wherein the aminoglycoside antibiotic is one of a plurality ofdifferent antibiotics, and the delivering step is effected by deliveringthe antibiotics in periodic dosages of different subsets of theantibiotics.
 9. The method of claim 1, wherein the aminoglycosideantibiotic is one of a plurality of different antibiotics, and thedelivering step is effected by delivering the antibiotics in periodicdosages of different subsets of the antibiotics, wherein the differentantibiotics are aminoglycoside antibiotics.
 10. The method of claim 1,wherein the aminoglycoside antibiotic is one of a plurality of differentantibiotics, and the delivering step is effected by delivering theantibiotics in periodic dosages of different subsets of the antibiotics,wherein the method further comprises introducing into the gut aneffective amount of probiotic, gut-beneficial microbial cultures inperiodic dosages.
 11. The method of claim 1, wherein the determiningstep is done by detecting an indication of or predisposition to polypsor colorectal cancer.
 12. The method of claim 1, wherein the deliveringstep is effected by delivering a constant over time dosage of theaminoglycoside.
 13. The method of claim 1, wherein the delivering stepis effected by delivering a varying over time dosage of theaminoglycoside.
 14. The method of claim 1, wherein the method furthercomprises introducing into the gut an effective amount of a probiotic,gut-beneficial microbial culture.
 15. The method of claim 1, wherein thegut-beneficial culture comprises a microbe selected from the groupconsisting of Lactobacillus, Bifidobacteria, Bacteroides, Streptococcus,and Saccharomyces.
 16. The method of claim 1, wherein the gut-beneficialculture comprises a microbe selected from the group consisting of: L.acidophilus, L. reuteri, L. acidophilus, L. bulgaricus, L. plantarum, L.casei, L. gasseri, L. GG, S. thermophilus, S. salivarius, B. bifidus,and Saccharomyces boulardii.